体外传代培养的角膜缘干细胞特性的研究  

Study on characteristics of cultured limbal stem cells in vitro

作　　者：杜仪琴[1] 陈静[2] 朱秀安[1] 李凌松[2] 

机构地区：[1]北京大学第三医院北京大学眼科中心,100083 [2]北京大学干细胞研究中心

出　　处：《眼科研究》2006年第4期389-392,共4页Chinese Ophthalmic Research

摘　　要：目的探讨体外培养的角膜缘干细胞的特性并寻找其表面标志。方法体外扩增人角膜缘干细胞,检测第4代培养细胞Brd-U掺入率及p63、connexin43、角蛋白3/12的表达情况,分析细胞表面分子的表达。结果培养细胞Brd-U24h掺入率明显低于6d掺入率;第4代细胞表现为p63单阳、p63-connexin43双阳和角蛋白3单阳的混合体,而角膜上皮细胞只表达角蛋白。流式细胞分析传代的干细胞β4integrin,CD90(+),α6integrin,CD14(-),角膜上皮细胞CD14(+)。结论体外传代培养的角膜缘干细胞部分保持干细胞特性,具有增生能力。p63是增生细胞的标志。β4integrin+/α6integrin-联合CD90+/CD14-可能纯化角膜缘干细胞。Objective To investigate the maintaining of properties passaged limbal stem cells in vitro and seek the specific surface markers of limbal stem cells. Methods Human corneal limbal stem cells from donor were cultured and passaged in DMEM/F12 plus supplements. BrdU labeling index of passaged cells in 24 hours and 6 days was detected, p63,keratin 3,keratin 12 and connexin43 were detected on the fourth passage limbal stem cells and corneal epithelial cells by RT-PCR,western blot and immunofluorescent staining. Surface proteins of these two cells were analyzed using flow cytometry technique. Results The BrdU labeling index of passaged cells in 24 hours was much lower than that in 6 days. RT-PCR results showed both p63 and keratin 12 were expressed in passaged limbal stem cells and only keratin 12 was detected in corneal epithelial cells. Similar results were seen in the protein expression by western blot technique. Immunofluorescent results revealed that the passaged cells were positive stained for p63, for both p63 and connexin43 or positive for keratin 3, respectively. Flow cytometry determined that passaged limbal stem cells were positive for [34 integrin and CD90, but were negative for ct6 integrin and CD14,while corneal epithelial cells were positive for CD14. Conclusion Passaged limbal stem cells could partly keep their property of stem cells and have a potential proliferation ability, p63 is the marker of proliferation cells. β4 integrin ＋ /α6 integrin-plus CD90 ＋/ CD14-may be useful to isolating and purifying limbal stem cells.

关 键 词：角膜缘干细胞 体外培养 P63 角蛋白 细胞表面分子 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]